Galactose oxidase (E.C. 1.1.3.9, GOase) is a copper-containing extracellular enzyme which oxidizes the primary hydroxyl groups of many alcohols and of galactose. Although the enzyme is produced by a number of fungus species, fermentation of Dactylium dendroides is currently the best practical source. A simple method for the growth of this fungus and purification of the excreted enzyme from the growth medium is described by Tressel and Kosman, Analytical Biochemistry , 105. 150-153 (1930).
In this process the enzyme is grown in a fungal culture aerobically in the dark for several days at 20.degree. C. This fungus is then transferred to a glucose-based liquid medium and grown aerobically for about 2 days at 20.degree. C. A completely artificial medium is used; it is a mixture of sorbose, glucose, traces of metal ions as micronutrients, and thiamine as the necessary vitamin. Growth of the culture takes usually 5 to 7 days. Isolation of the galactose oxidase involves a number of steps which begins with precipitation in the presence of microcrystalline cellulose. The purification is completed by chromatography on a phosphocellulose column.
The enzyme contains 1 atom of copper and the presence of the cupric (Cu .sup.+2 ions is necessary for enzymic activity. When the galactose oxidase is grown under conditions of copper deprivation, Dactylium dendroides synthesizes and excretes a catalytically inactive protein, aplagalactose oxidase. Catalytic activity of the protein appears when cupric ion is added to the copper depleted solution (see, Shatzman and Kosman, Biochim. Biophys. Acata. 1978, 544, 163-169 and Markus et al. , G. Avigad. Appl.. Microbiol., 13 (5), 686-693 (1965)). This result implies that although copper is necessary for the catalytic activity of galactose oxidase, cupric ion neither induces synthesis nor is necessary for the complete assembly and secretion of proteins by Dactylium dendroides. On the other hand, Japanese workers (Aisaka and Terada, Agric. Bio. Chems., 45, 2311-2316 (1981)) concluded that the synthesis of galactose oxidase protein by Gibberella Fujikuroi is a phenomenon regulated by copper.
Copper ion also preserves enzyme activity in unpurified fermentation medium by preventing complexation of galactose oxidase with an inhibitor (Avigad and Markus, Israel J. Chem., 3, 193 (1966)). It is known that Dactylium dendroides produces at least one galactose oxidase inhibitor identified as a heptapeptide. This protein forms a stable inactive complex with galactose oxidase in the absence of copper. Presence of 1 to 10 mM cupric ion not only prevents this inhibition but also causes slow activation of the inhibited enzyme. The inhibiting components are usually removed from galactose oxidase preparations by chromatography.
The present process produces about a 45% yield of enzyme (measured as recovered activity) over a three day isolation period. It has now been found that this isolation and purification can be simplified and conducted on large quantities of enzyme preparation mixtures by the use of ultrafiltration and high performance liquid chromatography purification techniques. Gram quantities of the galactose oxidase can be prepared in a day.
Accordingly, it is an object of this invention to prepare large quantities of galactose oxidase in yields of above 70% (measured as recovered total activity) in an efficient manner.
All percentages are by weight unless otherwise indicated.